Human fibroblast switches to anaerobic metabolic pathway in response to serum starvation: a mimic of warburg effect.

Fibroblasts could be considered as connective tissue cells that are morphologically heterogeneous with diverse functions depending on their location and activity. These cells play critical role in health and disease such as cancer and wound by Production of collagen, fibronectin, cytokines and growth factors. Absence of insulin and other growth factors in serum deprivation condition and similarity of this condition to the environment of tumor cells and ulcer made us to investigate anaerobic glycolysis in these cells. To this end, we cultured fibroblasts isolated from fresh human newborn foreskin in serum free medium for 16, 24, 48 and 72 hrs and measured glucose consumption, lactate secretion and intracellular LDH in these cells. The results showed despite the lack of insulin, the 16hr serum starved fibroblasts consumed glucose similar to non-starved fibroblasts control. Moreover, in this condition these cells secreted higher levels of lactate and exhibited higher levels of intracellular LDH in comparison to non-starved fibroblasts control. Thus it could be concluded that in serum starvation condition, the newborn human dermal fibroblasts may change the metabolic strategy to Warburg effect. This finding opens a new perspective to further understanding the basic mechanisms involved in communication between tumor cells and fibroblasts.

epithelial cell proliferation and differentiation by paracrine signaling pathways (9)(10), moreover, the bilateral relation between cancer cells and fibroblasts surrounding the tumor plays a role in tumor survival (11)(12) Many studies have shown low-nutrient environments are commonly found in the region of tumor and wound and have an important role in gene expressions, angiogenesis, metabolism, and etc., in this condition, cancer associated fibroblasts help cancer cells to growth and metastasis (13)(14)(15)(16). Cancer cells also induce anaerobic glycolysis in their associated fibroblasts called reverse Warburg effect (11,(17)(18). These mechanisms can cause tumor recurrence, metastasis, and drug resistance in all types of human breast cancer (19)(20).
Our previous findings showed that serum starved fibroblast exhibits significantly a higher proliferation rate more than the non-starved control after re-feeding (21). The absence of insulin and other growth factors in this condition and the similarity to the environmental condition of fibroblasts surrounding tumor cells and ulcer, made us to investigate anaerobic glycolysis in these cells.

Materials and Methods
Human newborn dermal fibroblast isolation and culture Isolated fibroblast based on Pandamoz et al.'s method (22), from six human newborn foreskins at 1-3 months of age that underwent routine circumcision during January 2012 to June 2012 in Amirkola Children Hospital, Babol/ Iran.

Statistical analysis
Data were represented as mean±SD. Statistical analysis was performed using two-sample, unpaired

Cytoplasmic spreading and cell shape changes in starved fibroblast
As it can be obviously seen in figure 1 after 24 hr, the starved fibroblast progressively spread its cytoplasm and consequently changed its shape probably to adapt to metabolically unfavorable condition induced by serum starvation. In figure 1, nonstarved and the 6, 16 hr starved fibroblast have spindle shape and distinct border. After 24 hr starvation, the more serum starvation lasts, the more cytoplasmic surface will be wide and their borders will be indistinct but the cells are shiny and alive even up to 72 hrs serum starvation.

Starved fibroblast has high glucose consumption despite the absence of insulin
We were not able to detect insulin at a

level of intracellular LDH Increased during serum starvation
The level of lactate dehydrogenase (LDH) in starved and non-starved fibroblast cell lysate was determined using routine clinical biochemistry laboratory method. The data showed (Fig 3) lactate  it was twenty-fold more than the non-starved control.

Serum starved fibroblasts increased lactate production
The measurement of lactate level in starved fibroblast cell culture supernatant showed (Fig 4-A) the significant level of lactate detected in starved fibroblast culture supernatants at each time point.
As shown in figure 4-B, there is a direct correlation between intracellular LDH and secreted lactate in starved fibroblast with R 2 =0.67.

Discussion
Increased lactate secretion by fibroblast  Our other observation showed newborn serum starved fibroblasts may do "Warburg effect". The cancer cells even with enough oxygen supply select the anaerobic glycolysis pathway for supplying energy that called Warburge effect (24)(25). In our study, it seems that the starved cells selected anaerobic glycolysis pathway for energy production despite of the presence of enough oxygen.
Previously, it has been shown that the cancerassociated fibroblasts also produce lactate through Warburg effect that leads to growth and tumor cell survival even in the absence of angiogenesis (11)(12)